Doubly telecentric lens and imaging system for multiwell plates

ABSTRACT

A multiple element color-corrected doubly telecentric lens and imaging system useful for imaging multiwell plates is described. The lens contains a biconvex field lens element L 1,  a positive meniscus lens element L 2,  concave toward the incident light side, a double-Gauss lens element group, a positive meniscus lens element L 10,  convex toward the incident light, a positive meniscus lens element L 11,  convex toward the incident light, and a plano concave field flattener lens element L 12,  concave toward the incident light side. The lens is very sensitive, and can be used to image scintillation proximity assays in multiwell plates.

FIELD OF THE INVENTION

The present invention relates to the imaging of multiwell plates, inparticular, to the use of a doubly telecentric lens and system foraccurate and efficient detection of low levels of light from highdensity multiwell plates.

BACKGROUND OF THE INVENTION

Microtiter or multi-well plates are becoming increasingly popular invarious chemical and biological assays. Further, high-density formatplates, such as 384, 864 and 1536 well plates, are beginning to displace96-well plates as the plate of choice. Many of the assays conducted inmultiwell plates employ some type of light detection from the plate asthe reporter for positive or negative assays results. Such assaysinclude fluorescence assays, luminescence assays (e.g., luciferase-basedassays), phosphorescence assays, scintillation assays, and the like. Inparticular, with the advent of solid phase scintillating materials,capsules and beads, homogeneous scintillation proximity assays (SPA) arenow being performed with increasing frequency in multiwell plates.

Detection of light signals from multiwell plates in the past hastypically been done using plate readers, which generally employ aphotodetector, an array of such photodetectors, photomultiplier tubes ora photodiode array to quantify the amount of light emitted fromdifferent wells. Such plate readers have been disclosed, for example, byRussell, et al., U.S. patent Ser. No. 4,810,096, issued Mar. 7, 1989,and VanCauter, et al., U.S. patent Ser. No. 5,198,670, issued Mar. 30,1993. Although plate readers can detect the total light from each well,they have a number of limitations. For example, plate readers aretypically not capable of resolving discrete light sources in a singlewell, so they could not be used, for example, to differentiate lightfrom different beads in one well. Further, most plate readers have fewerphotodetectors than there are wells in the plate, so at least some wellsmust be read serially, adding to the time required to complete theassays. This becomes a substantial problem in assays where the lightsignal is so low that each well needs to be in the detection field foran extended period of time (e.g., tens of minutes). In addition, mostcurrently-available plate readers have been designed for 96-well plates.Although some can be adapted for, e.g., 384-well plates, the adaptationdoes not result in any significant increase in throughput, since a384-well plate going through a modified 96-well reader typically takesfour times as long to read as a 96-well plate.

Another technique that has been applied to the detection of light frommultiwell plates is imaging. Prior art imaging systems typicallycomprise a standard 50-55 mm f1.4 photographic lens coupled to a camera.While such systems can be used to image an entire multiwell plate, andtheoretically provide resolution of discrete light points withinindividual wells, they have poor sensitivity, even when coupled toefficient cameras, so that many assays still require imaging times oftens of minutes or more. Other assays, such as SPA bead-based assays,cannot be performed at all due to lack of sensitivity. Further, imagesacquired with such systems suffer from vignetting and lateral distortioneffects, making it difficult or impossible to compare signals fromcenter portions of the plate with signals from lateral wells.

The present invention provides lenses and systems which overcome theabove-described disadvantages of prior art methods of light detection inmultiwell plates. In particular, the present invention provides, for thefirst time, a doubly telecentric lens-based system with the ability toimage SPA bead-based assays. The telecentric lens of the invention iseconomical to manufacture due to a design employing a minimal totalnumber of lens elements, the use of spherical lens elements, andgenerous tolerance limits in lens fabrication. Further, the telecentriclens of the present invention is the first such lens to provide anunprocessed image of a multiwell plate that is substantially free fromvignetting, chromatic aberration and distortion.

SUMMARY OF THE INVENTION

In one aspect, the present invention includes a multiple element doublytelecentric lens for imaging a substantially flat object onto an imageplane. The lens is preferably color-corrected, particularly in the rangeof 400 nm-700 nm, and comprises, in succession from a side of incidentlight, the following lens elements or groups (illustrated in FIGS. 2 and3): (i) a biconvex field lens element L1, (ii) separated by a distance Dfrom the field lens element, a positive meniscus lens element L2,concave toward the incident light side, (iii) a double-Gauss lenselement group, (iv) a positive meniscus lens element L10, convex towardthe incident light, (v) a positive meniscus lens element L11, convextoward the incident light, and (vi) a plano concave field flattener lenselement L12, concave toward the incident light side. Changing distance Dchanges magnification of the telecentric lens.

In a preferred embodiment, the double-Gauss lens element groupcomprises, in succession from the side of incident light, (i) anincident-side lens element group, comprising, in succession from theside of incident light, (a) a biconvex lens element L3, (b) a positivemeniscus lens element L4, convex toward the incident light, and (c) adoublet formed of two positive meniscus lens elements, L5 and L6, bothconvex toward the incident light; (ii) an aperture stop; and (iii) animage-side lens element group, comprising, in succession from the sideof incident light, (a) a doublet formed of a biconcave lens element L7and a biconvex lens element L8, the doublet being concave toward theincident light, and (b) a biconvex lens element.

The adjacent surfaces of the above-listed lens elements are preferablyseparated by distances as follows: between about 11.4″ and about 13.4″between element L1 and element L2; about 3.9″ between element L2 andelement L3; about 0.3″ between element L3 and element L4; about 0.04″between element L4 and element L5; about 0.0″ between element L5 andelement L6; about 2.3″ between element L6 and element L7; about 0.0″between element L7 and element L8; about 0.1″ between element L8 andelement L9; about 0.1″ between element L9 and element L10; about 0.1″between element L10 and element L11; and about 0.3″ between element L11and element L12.

Further, the lens described above preferably has the followingcharacteristics, where elements L1-L12 are made of glass and havesurfaces S1-S24; each of the surfaces is convex (CX), concave (CC) orplano (XX); and the CX and CC surfaces have a radius measured in inches:

Element Glass Surface Radius CX/CC L1 Schott BK7 S1  30.008″  CX (No.517642) S2  62.327″  CX L2 Schott BK7 S3  88.185″  CC (No. 517642) S4 18.483″  CX L3 Schott LaKN22 S5  28.992″  CX (No. 651559) S6  19.682″ CX L4 Schott LaKN22 S7  6.938″ CX (No. 651559) S8  26.500″  CC L5 SchottLaFN28 S9  2.644″ CX (No. 773496) S10 3.024″ CC L6 Ohara PBH53 S113.024″ CX (No. 847239) S12 1.564″ CC L7 Ohara PBH53 S12 1.836″ CC (No.847239) S14 14.212″  CC L8 Schott LaFN28 S15 14.212″  CX (No. 773496)S16 2.776″ CX L9 Ohara LAL14 S17 6.589″ CX (No. 697555) S18 8.284″ CX L10 Ohara LAL14 S19 2.798″ CX (No. 697555) S20 10.465″  CC  L11 SchottBK7 S21 5.195″ CX (No. 517642) S22 7.752″ CC  L12 Ohara LAH53 S23 7.236″CC (No. 806410) S24 PLANO XX

In another embodiment, the telecentric lens is designed to form afocused image at the image plane when the field lens element (L1) ispositioned between about 20 mm and about 30 mm, preferably about 25mmfrom the object. In other embodiments, distance D is preferably betweenabout 11.4″ and about 13.4″; the lens has a numerical aperture ofbetween about 0.5 and 0.6, preferably about 0.55; the lens has amagnification of between about −0.20 and −0.25, preferably about −0.224;and the lens has a field of view of between about 4″ and about 6″,preferably about 5″ in diameter.

In another aspect, the invention includes a multiple element telecentriclens for imaging a multiwell plate having square wells onto a CCD pixelarray. The lens has a magnification that results in each well of theplate mapping to an integer number of pixels in the CCD array. In oneembodiment, the CCD array is a 1024×1024 array. In a related embodiment,the multiwell plate has dimensions of a standard multiwell plate with a2:3 aspect ratio of rows:columns, and all wells in a single row areimaged onto a rectangular region of the 1024×1024 CCD array, the regionbeing 1008 pixels in length. In another embodiment, the multiwell plateis selected from the group consisting of a 384-well plate, an 864-wellplate, and a 1536-well plate.

Still another aspect of the invention includes a multiple element doublytelecentric lens for imaging a substantially flat object onto an imageplane. The lens is preferably color corrected and has the followingcharacteristics: (i) between 12 and 16 lens elements, preferably 12;(ii) a numerical aperture of between about 0.5 and 0.6, preferably 0.55;(iii) less than about 5% vignetting, preferably substantially zerovignetting; (iv) less than about 0.05% distortion; (v) a magnificationof between about −0.20 and about −0.25, preferably between about −0.22and about −0.23, more preferably about −0.225; and (vi) a field of viewof between about 4″ and about 6″, preferably about 5″ in diameter.

In one embodiment, the telecentric lens is used for imaging a multiwellplate having square wells onto a CCD pixel array, and has amagnification that results in each well of the plate mapping to aninteger number of pixels in the CCD array.

Also included as part of the invention is a system for imaging a platehaving a plurality of wells. The system comprises: (i) a multipleelement telecentric lens suitable for imaging multiwell plates (e.g., asdescribed above), (ii) a camera operably connected to the lens, and(iii) a robot having a base member and at least one arm, wherein the armincludes a grasping mechanism which is adapted to grasp the plate, andwherein the grasping mechanism is configured to receive the plate in arepeatable and known location such that the location of each wellrelative to the grasping mechanism is known by the robot.

In a related aspect, the invention includes a system for imaging astandard sample plate. The system includes a multiple element doublytelecentric lens as described above, a camera operably connected to thelens, and a chamber for receiving the plate during imaging. In oneembodiment, the camera is a cooled 1024×1024 CCD array camera. Inanother embodiment, the telecentric lens and the camera are mounted on aslide support such that magnification of the lens and focus of thecamera can be adjusted separately.

In yet another embodiment, the system further comprises a means forpositioning the plate in the chamber, such as a robot, conveyer belt, orthe like. A system using a robot preferably further comprises aplurality of stations at known locations relative to the robot, andfurther includes a processor associated with the robot, where theprocessor is configured to control movement of the robot to place theplate at predetermined locations at the stations. The standard sampleplate preferably has having a plurality of wells, the robot preferablyhas a base member and at least one arm, the arm preferably includes agrasping mechanism which is adapted to grasp the plate, and the graspingmechanism is preferably configured to receive the plate in a repeatableand known location such that the location of each well relative to thegrasping mechanism is known by the robot.

In still another embodiment, the system further comprises a translationmechanism for axially translating the frame member, wherein the graspingmechanism comprises a frame member having edges which are adapted toframe at least a portion of a periphery of the plate in a repeatable andpredictable manner so that the location of the wells of the platerelative to the frame member is known when the plate is received intothe frame member; and a clamping arm that is pivotally attached to theframe member and which is adapted to engage a portion of the peripheryof the plate to secure the plate to the frame member when the plate isreceived within the frame member.

The invention further includes a method for imaging a multiwell plate.The method comprises the steps of (i) positioning the plate under amultiple element doubly telecentric lens such as is described above,(ii) collecting light from the plate with the telecentric lens, (iii)transmitting the light through the lens to an image detection device,and (iv) using output from the image detection device to generate animage of the multiwell plate. In one embodiment, the image detectiondevice is a CCD camera.

Also part of the invention is a method for imaging a solid-phasescintillant used in a scintillation proximity assay (SPA) in a standardmultiwell plate. The method includes the steps of (i) positioning theplate under a multiple element telecentric lens suitable for imaging themultiwell plate, for example, a lens such as is described above, (ii)collecting light from the plate with the telecentric lens, (iii)transmitting the light through the lens to an image detection device,and (iv) using output from the image detection device to generate animage of the multiwell plate. In one embodiment, the image detectiondevice is a (preferably cooled) CCD camera. dr

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIGS. 1A and 1B are cross-section schematic diagrams of light rays fromwells of a multiwell plate passing through a non-telecentric lens (FIG.1A) and a telecentric lens (FIG. 1B) to a CCD camera.

FIG. 2 is an illustration of a color-corrected doubly telecentric lensin accordance with a first embodiment of the invention.

FIG. 3 is a cross-sectional view of a lens assembly containing lenselements illustrated in FIG. 2.

FIG. 3A is an illustration of a shutter suitable for use with thepresent invention.

FIG. 4 is a side view of an imaging system comprising a telecentric lensof the invention mounted on a lens support.

FIG. 4A is a perspective view of a slide used to attach a telecentriclens of the invention to the lens support illustrated in FIG. 4.

FIG. 5 is a perspective view of an imaging system comprising a robot forholding plates during imaging in accordance with the present invention.

FIG. 6 is an exploded perspective view of an exemplary graspingmechanism of a robot used in one embodiment of an imaging system of theinvention.

FIG. 7 is a top view of the grasping mechanism of FIG. 6.

FIG. 8 is a side view of the grasping mechanism of FIG. 6.

FIG. 9A is a perspective view of an exemplary robot suitable for use inan imaging system of the invention.

FIG. 9B is a more detailed view of the robot of FIG. 9A taken alonglines B—B.

FIGS. 10A and 10B illustrate movement of the robot of FIG. 9A.

FIG. 11 is a perspective view of a patterned shelf of one of the racksof the system of FIG. 5.

FIG. 12 is a computer-generated image of SPA bead luminescence producedusing an imaging system of the invention as described in Example 1.

FIG. 13 shows binding inhibition curves and IC₅₀ values obtained fromthe data shown in FIG. 12.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

I. Definitions

Unless otherwise specified, terms relating to optics are understood tohave the meanings accorded to them by those of skill in the art ofoptics or photonics. Such meanings can be found, for example, in the“Photonics Dictionary”, 40^(th) International Edition, Laurin PublishingCo., Pittsfield, Mass. (1994); and/or in the “Handbook of Optics, 2^(nd)Ed.”, M. Bass, Ed., McGraw-Hill, Inc., 1995, both of which are hereinincorporated by reference.

The term “lens element” refers to a simple lens formed of a single pieceof optical glass.

The term “optical element” refers to an object which modifies acharacteristic of light along an optical path when the optical elementis placed in that optical path. Examples of optical elements includelens elements, filters, polarizers, diffraction gratings, lens stops,shutters and the like.

The terms “compound lens” and “multiple element lens” refer to a lenscomposed of a plurality of lens elements that may or may not be cementedtogether.

A “standard multiwell plate” is a multiwell plate that is between about4.5″ and about 5.5″ in length at its longest point, and between about 3″and about 3.75″ in width at its widest point. The dimensions of anexemplary standard multiwell plate are about 5″ in length and about3.38″ in width. Standard multiwell plates can have any number of wells.A commonly-used multiwell plate has 96 wells in an 8×12 format at ˜9 mmcenters. Other multiwell formats suitable for use with the presentinvention include, but are not limited to, 384, 864, 1536-well formats.Standard multiwell plates have a 2:3 aspect ration of rows:columns, sothat, e.g., an 864-well plate has 864 wells arranged in 24 rows and 36columns. An exemplary standard 864 well plate which may be used with theinvention is described in co-pending U.S. application Ser. No.08/868,689, filed Jun. 4, 1997, the disclosure of which is hereinincorporated by reference.

A “standard sample plate” has the dimensions and characteristics of astandard multiwell plate as defined above, except that the definitionencompasses multiwell plates as well as plates having only a single well(e.g., “omni plates”).

II. Overview of Invention

The present invention describes an imaging system suitable for resolvingpoints separated by about 140 μm or more. The system is particularlysuitable for accurately measuring the light from discrete regionsseparated by physical barriers, where the barriers around a particularregion limit the view of that region from points other than thosedirectly above the region. In a preferred embodiment, the system has anoptical field with a diameter of between about 4 and about 6 inches(preferably about 5 inches).

In one particular embodiment, described in detail below, the system isoptimized for the imaging of optical signals from wells of multiwellplates, particularly high-density multiwell plates such as 384-well,864-well or 1536-well plates. In an exemplary embodiment, the system isconstructed around a doubly telecentric color corrected lens having afield of view wide enough to image all the wells of a standard multiwellplate onto an image capture device, such as a charge coupled device(CCD) array of a CCD camera. Experiments conducted in support of thepresent invention demonstrate that a lens constructed as detailed hereinis at least about 7 times more sensitive than a standard non-telecentricsingle lens reflex (SLR) 50 mm f1.4 camera lens connected to the sameCCD camera. As such, a lens of the invention can be used to reduce thetime for a light-detection assays (e.g., a luminescence assay) by afactor of at least 7. Alternatively or in addition, the lens can be usedto conduct experiments with smaller amounts of reagents (producing lowerlevels of light) than was previously possible.

The increased sensitivity is made possible by a high numerical aperture(0.55) and by a design that uses only 12 lens elements (24 surfaces).The high NA allows for the collection of a wide cone of light from theobject, while limiting the number of optical elements limits light lossdue to reflection at each surface (even a multilayer coated surfacetypically reflects approximately 0.5% to 1% of the incident light).

Some advantages of a telecentric lens are apparent in the schematicillustrations of FIGS. 1A and 1B. In multiwell plates with well wallsseparating individual wells, light coming from each well is partiallyblocked by the walls of that well. If all the light is effectivelygathered at a single central point above the plate, as is done using astandard non-telecentric lens (FIG. 1A), the central wells will beimaged more efficiently than the wells near the sides of the plate, inpart because some of the light from the side wells will be blocked bythe “inner” walls of the wells (those walls closest to the center of theplate), and in part because the detection field is not centered aboutthe perpendicular ray coming from the center of the side well. Thisresults in a “shadowing” effect caused solely by not using a telecentricimaging system, generating an image where wells in the center appearbrighter than those at the edges of the plate, even though all wellsemit the same amount of light. By using a telecentric lens having afield lens or lens element of a diameter that encompasses the entiremultiwell plate (FIG. 1B), such shadowing effects are eliminated.

A recognized benefit of telecentric lenses is that out-of-focus imageshave the same magnification as in-focus images. This characteristic hasled to wide use of telecentric lenses in applications requiring precisemeasurements, with no tolerance for changes in magnification ofout-of-focus images. In the present invention, uniform magnification ofout-of-focus images is important in reducing cross-talk between adjacentwells (mistakenly attributing the light originating from one well to aneighboring well). Cross talk results, for example, when an assay isdesigned to detect luminescence in bulk solution, and the wells of theassay plate each contain enough solution so that the solution surfaceand the well bottom are in different focal planes. The out-of-focuslight signals detected with a non-telecentric lens in such situationscan obscure the walls of the wells (i.e., well boundaries) in an imageof the plate, making quantification of signal in different wellsdifficult or impossible.

Full realization of the above advantages requires the use of a doublytelecentric lens—that is, a lens that is telecentric at both the objectand image planes. On the object side, telecentricity provides foruniform pick up of light from the wells over the entire field, i.e.,each well is seen identically. On the image side, the telecentricityserves two purposes: one is to get the high aperture (e.g., NA 0.55)cone of light into the CCD array without vignetting, and the other is toallow the image position on the CCD to remain constant even though partsof the image are defocused as a result of imaging different depths inthe wells as described above.

An aspect of double telecentricity is that the magnification is notadjustable by moving the whole lens and image plane with respect to theobject. For this reason, a preferred embodiment of the telecentric lensof the invention employs an object field lens (element L1 as describedbelow) that remains fixed relative to the object, while the remainder ofthe lens moves during changes in magnification. Changes in focus areaccomplished by moving the camera or detection device relative to theimage plane of the lens, rather than changing the position of lenselements relative to the object plane or relative to one another. Sincethe lens is doubly telecentric, the paraxial focal length is inprinciple infinite. However, an effective focal length (EFL) can beattributed to portions of the doubly telecentric lens. In a preferredembodiment, the field lens (L1) has an EFL of about 995 mm, while theremainder of the lens has an EFL of about 201 mm.

A doubly telecentric lens of the invention is preferably designed to becolor-corrected and to have zero vignetting. Color correction enablesthe use of multiple wavelength light without loss of resolution ordegradation of the image due to chromatic aberration. Elimination ofvignetting enables the accurate quantification of light from differentregions of the multiwell plate being imaged. An additional benefit of novignetting combined with double telecentricity is the uniformity ofillumination. Even in the absence of vignetting, there is ordinarily abrightness fall off with field known as the cosine fourth law, where thearc cosine is the angle of the chief ray in object and image space. Witha doubly telecentric system, both the object and image chief rays areessentially zero, so the cosine to the fourth power is ˜1.

Further, the lens preferably has a distortion of less than about 0.05%.This translates to less than a 3 μm error between the center and edgesof the field at the image plane, which is about {fraction (1/10)} of theresolution of a typical 1024×1024 CCD array (˜28 pm per pixel), such asis used in an exemplary embodiment of an imaging system of theinvention.

III. Doubly Telecentric Lens

FIG. 2 shows the optical elements of a multiple element or compounddoubly telecentric lens 10 according to a preferred embodiment of thepresent invention. Lens 10, designed to minimize spherical (less thanabout 0.15%) and chromatic aberration, has a distortion of less thanabout 0.05%, substantially zero vignetting, a numerical aperture (NA) of0.55, and a magnification of between about −0.20 and −0.25. Lens 10 isformed of twelve individual lens elements, L1-L12. The elements arenumbered from left to right in FIGS. 2 and 3, with element L1 beingadjacent object plane OP, and element L12 being adjacent image plane IP.Elements L3-L9 form a “double Gauss” lens element group 12, withelements L3-L6 forming an incident side lens element group 14, andelements L7-L9 forming an image-side lens element group 16. Each lenselement has two surfaces, which are indicated for the 12 elements byS1-S24. Lens 10 is shown positioned about optical axis AX. Also shownare ray tracings starting from points OP1, OP2 and OP3 in object planeOP and continuing through lens 10 to points IP1, IP2 and IP3,respectively, in image plane IP. In between incident side lens elementgroup 14, and image-side lens element group 16, is an aperture stop AS,positioned at the pupil of the lens.

Physical characteristics of individual lens elements used to make apreferred embodiment of the invention are provided in Table 1, below.The characteristics include the center thickness (C.T.) in inches, theouter diameter (O.D.) in inches, the type of glass, an opticalcharacteristic number, the refractive index, and the dispersion.

TABLE 1 ref Element C.T. O.D. Glass Number index disp L1 0.7087 6.000Schott BK7 No. 517642 1.517 64.2 L2 0.7874 7.500 Schott BK7 No. 5176421.517 64.2 L3 0.9055 7.040 Schott LaKN22 No. 651559 1.651 55.9 L4 0.98437.000 Schott LaKN22 No. 651559 1.651 55.9 L5 1.2295 4.880 Schott LaFN28No. 773496 1.773 49.6 L6 0.3232 4.000 Ohara PBH53 No. 847239 1.847 23.9L7 0.2232 4.750 Ohara PBH53 No. 847239 1.847 23.9 L8 1.5071 3.860 SchottLaFN28 No. 773496 1.773 49.6 L9 1.3032 4.750 Ohara LAL14 No. 6975551.697 55.5  L10 1.063  4.140 Ohara LAL14 No. 697555 1.697 55.5  L110.9843 4.140 Schott BK7 No. 517642 1.517 64.2  L12 0.1969 2.756 OharaLAH53 No. 806410 1.806 41.0

Lens element L1 comprises surfaces S1 and S2, with S1 facing the objectplane, lens element L2 comprises surfaces S3 and S4, and so on.Additional information, broken down by the individual surfaces of eachelement, is provided in Table 2, below. The information includes (i) theradius of each surface in inches; (ii) whether the surface is convex(CX), concave (CC) or PLANO (XX); and (iii) the clear aperture diameterof each surface in inches. Also shown in Table 2 are the distances (ininches) between adjacent surfaces of the individual lens elements orbetween lens element surfaces and the aperture stop.

TABLE 2 Surface Distance Radius CX/CC Clear Aperture S1  30.008  CX5.570 S2  62.327  CX 5.620 12.413  range 11.4 to 13.4 S3  88.185  CC6.880 S4  18.483  CX 6.930 3.877 S5  28.992  CX 6.691 S6  19.682  CX6.638 0.320 S7  6.938 CX 6.057 S8  26.500  CC 5.768 0.039 S9  2.644 CX4.594 S10 3.024 CC 3.673 0.000 S11 3.024 CX 3.673 S12 1.564 CC 2.7501.538 Aperture Stop 0.735 S13 1.836 CC 2.322 S14 14.212  CC 3.013 0.000S15 14.212  CX 3.013 S16 2.776 CX 3.739 0.096 S17 6.589 CX 4.376 S188.284 CX 4.375 0.120 S19 2.798 CX 3.891 S20 10.465  CC 3.487 0.082 S215.195 CX 3.200 S22 7.752 CC 2.236 0.280 S23 7.236 CC 1.990 S24 PLANO XX1.820

In an exemplary embodiment, lens elements L1-L12 were manufactured usingthe glasses listed in Table 1 according to the following commonspecifications: The glasses used were striae grade A having ahomogeneity of +/−5e-6; fine annealed to </=10 nm/cm. The surfacesphericity tolerance was ¼ wave at 633 nm, with a scratch & digtolerance of 20/10. The thickness (CT) tolerance was +/−0.002″, and theouter diameter (OD) tolerance was +0.000″, −0.0009″. Centrationtolerance was 5 μm TIR on all edges and flats. Doublets were cementedusing “UV” cure optical cement (Summers Labs P-92) followingmanufacturer's instructions.

Lens elements L1-L4 and L9-L12 are all airspaced singlet elements.Elements L5 and L6 form a first doublet, and elements L7 and L8 form asecond doublet. Field lens L1 is a biconvex lens positioned betweenabout 20 mm and about 30 mm (typically about 25 mm) from the object(e.g., multiwell plate) that is being imaged. Lens element L2 is ameniscus lens element oriented concave toward the incident light side,and is spaced from lens element L1 by distance of about 11.4″ to about13.4″ along optical axis AX. The purpose of element L2 is to convergethe light toward aperture stop AS, with the effect of decreasing thediameter of the lens elements between L2 and aperture stop AS.

Lens elements L3-L9 form a double Gauss lens element group, orobjective, centered about aperture stop AS. The double Gauss objective,also referred to as a Biotar objective, is described in U.S. Pat. No.2,117,258, issued in 1938. In its basic form, a double Gauss lenselement group employs two negative-meniscus inner doublets and twosingle positive outer elements. Other forms employ additional elements,as is described, for example, in Chapter 17 of “Modern Lens Design—AResource Manual” by W. J. Smith (McGraw-Hill, Inc., New York, 1992). Theexemplary embodiment described herein uses two positive outer elements(L3 and L4) on the incident light side in place of the single outerelement used in the basic double Gauss design, to provide for improveddistortion characteristics and correction of spherical and chromaticaberration.

As seen in FIGS. 2 and 3, element L3 is a biconvex lens; element L4 is apositive meniscus lens with the convex side facing the incident light;elements L5 and L6 form a positive meniscus doublet with the convex sidefacing the incident light, and the concave side adjacent aperture stopAS. Elements L7 and L8 are biconcave and biconvex, respectively, whichform a doublet with the concave side facing aperture stop AS and theincident light. Element L9 is a biconvex lens. Elements L10 and L11 arepositive meniscus lenses with their convex sides facing the incidentlight. Element L12 is a negative field flattener lens for correctingresidual field curvature.

The lens elements comprising telecentric lens 10 are all preferablycoated with a broad band, low scatter coating optimized for 450-650 nmlight: <=½% 450-650 nm, with ¼% goal emphasized for 450-550 region. Anexemplary low scatter multi-layer coating with these characteristics isavailable from, e.g., Thin Film Labs, Milford, Pa. Since the lenspreferably uses high-index glass, surfaces to be cemented in doubletsare also coated before cementing to match to the lower index opticalcement.

Functional characteristics of the lens include the following: The lens(i) can image all the wells of a standard multiwell platesimultaneously, i.e., has a field of view that is between about 4 andabout 6 inches (preferably about 5 inches) in diameter, (ii) is colorcorrected over 450-650 nm (usefully corrected over 400-700 nm), (iii)has no vignetting, and (iv) can image objects within its field of viewonto an area of about 1.1″ in diameter. As such, the lens is optimizedfor imaging into a CCD camera having a CCD array on the order of about0.75″ to about 1.5″ per side, preferably about 1″ per side.

FIG. 3 shows lens elements L1-L12 of FIG. 2 mounted in a lens assembly20. Lens assembly 20 includes a lower portion 22 and an upper portion24. Lower portion 22 has a slightly smaller diameter than upper portion24, which allows lower portion 22 to be slidably inserted into upperportion 24 in a telescoping manner, so that distance D between adjacentsurfaces of L1 and L2 can be varied without admitting external light atthe junction of lower portion 22 and upper portion 24. Distance D istypically varied between about 11.4″ and about 13.4″ to change themagnification of the lens from about −0.219 to about −0.230. When D isequal to about 12.413″, the magnification is about −0.224.

In applications employing an imaging device utilizing discrete pixelsfor image acquisition, e.g., a CCD camera, the magnification ispreferably set so that each square well in the multiwell plate is imagedonto an integer number of pixels in the imaging device (“integer pixelmapping”). In a preferred embodiment, the imaging system of theinvention employs a CCD camera with a 1024×1024 array of pixels, and themagnification is set so that an entire row of wells maps to arectangular region of the CCD array, where the region is 1008 pixels inlength and one well dimension in width. This particular arrangementconveniently allows integer mapping for square-well 384, 864 and1536-well plates having the standard 2:3 aspect ratio of rows:columns.For example, a standard 384-well plate has 16 rows and 24 columns.Setting the magnification so that an entire row (consisting of 24adjacent wells) maps to 1008 pixels provides that each well isrepresented by a 42×42 pixel region in the CCD array. In the case of an864-well plate having 24 rows and 36 columns, each well is representedby a 28×28 pixel region. Similarly, in a 1536-well plate having 36 rowsand 48 columns, each well maps to a 21×21 pixel region.

Setting the magnification (by adjusting D) so that each square well inthe multiwell plate is imaged onto an integer number of pixels in theimaging device as described above is advantageous in part because itaids in minimizing cross-talk between adjacent wells, and simplifiessubsequent digital analysis of the image. Specifically, if integermapping was used in the acquisition of an image of a multiwell plate,the image analysis software can be set up to automatically analyze thesignal from each well by allocating an integer number of pixels perwell. Further, in the case of 384 and 864-well plates, signals fromgroups of 4 pixels (2×2) can be “binned” in software (to reduce noiseand image file size while increasing sensitivity due to an increasedsignal to noise ratio) without losing the advantages gained by integerpixel mapping. Such binning results in a 21×21 “superpixel” array perwell for 384-well plates, and a 14×14 superpixel array per well for864-well plates.

Dimensions of lens assembly 20 are indicated by reference charactersd1-d11. An exemplary set of dimensions, suitable for making a lensassembly using lens elements dimensioned as detailed in Table 1, arepresented in Table 3, below.

TABLE 3 d_(n) Type Distance d1 length 12.57″  range: ˜11.6″-˜13.6″ d2length 17.29″  d4 length 9.39″ d5 length 0.85″ d6 diameter 9.12″ d7diameter 9.12″ d8 diameter 7.84″ d9 diameter 7.70″ d10 diameter 5.50″d11 diameter 4.71″

As illustrated in FIG. 3, a shutter 26 may be positioned between lenselements L6 and L7 at aperture stop AS (see FIG. 2) to control theamount of light passing through the lens. A shutter is desirable, forexample, when a telecentric lens of the invention is used to acquire animage with a camera, e.g., a film or CCD camera, to control film or CCDexposure. Positioning the shutter at aperture stop AS (FIG. 2) isadvantageous, since closing the shutter at this location in the opticalpath result in a gradual darkening of the entire image, as opposed to avisualization of the shutter screen moving across the image.

As best seen in FIG. 3A, an exemplary shutter 26 comprises shutter leafs28 slidably mounted on shutter guide rods 30, which in turn are fastenedto shutter frame 32. Also mounted on frame 32 via attachment members 33are air cylinders 34, each containing a piston 36. Distal ends ofpistons 36 are attached to shutter leafs 28 as illustrated. When aircylinders 34 are actuated by turning on an air or gas supply (notshown), pistons 36 are pushed out of cylinders 34 and act to separateshutter leafs 28, allowing light to propagate through a hole 38 inshutter frame 32, between object plane OP and image plane IP (FIG. 2).

IV. Multiwell Plate Imaging Workstation

In one aspect of the invention, the lens assembly described above ispart of an imaging system for imaging multiwell plates. As best seen inFIG. 4, imaging system 40 further includes a lens support 42, a camera44 operably connected to lens assembly 20, and a chamber 46 forenclosing the plate during imaging.

In the embodiment shown in FIG. 4, lens support 42 comprises an uprightsupport 48 and a base 50. Lower portion 22 of lens assembly 20 is boltedor clamped to base 50 at flange 52. Upper portion 24 of lens assembly 20is mounted to upright support 48 via two support arms, 54 and 56, whichare in turn fixed to a slide 58, such as LM Guide Actuator Type KR,manufactured by THK Co., LTD (Tokyo, Japan), and available from, e.g.,Bearing Engineers, Inc. (Redwood City, Calif.) as catalog numberKR4610AB-940L-01600. Slide 58 is attached to upright support 48.

As best seen in FIG. 4A, slide 58 comprises a support plate 60, tworails 62, a motor mount 64, and a ball screw 66. Slide 58 furthercomprises three slider blocks 68, 70 and 72, which slidably engageinside walls of rails 62. Slider block 68 engages ball screw 66 so thatrotation of ball screw 66 causes slider block 68 to translate alongrails 62. Slider blocks 70 and 72 are designed to freely translate alongrails 62 independent of any rotation of ball screw 66. Accordingly,until the slider blocks are otherwise linked together, slider block 68is the only block that necessarily moves when ball screw 66 is rotated.

Referring now to FIGS. 4, 4A and 5 together, it can be appreciated thatsupport arm 54 is attached onto slider block 68, and support arm 56 isattached onto slider block 70. In this manner, when imaging system 40 isassembled as shown in FIG. 4, rotation of ball screw 66 moves upperportion 24 relative to lower portion 22 of lens assembly 20, to achievea selected change in magnification. Support arm 56 together with sliderblock 70 follow passively, providing additional support for lensassembly 20. Ball screw 66 is controlled by a rotational stepper motor74, such as Stepping Motor M2-3437-S, manufactured by Intelligent MotionSystems (Marlborough, Conn.), and available from, e.g., BearingEngineers, Inc., Redwood City, Calif., as catalog number M2-3437-S.Activation of motor 74 turns ball screw 66 to either raise or lowerupper portion 24 as described above to achieve adjustments inmagnification. A power off brake (not shown), such as Model ID 1904 fromBearing Engineers, may be conveniently employed in series with motor 74to prevent ball screw 66 from turning when stepper motor 74 is notactivated. The brake can prevent both downward creep of the lens andstrain on stepper motor 74 during periods when motor 74 is notactivated.

Camera 44 is mounted immediately above lens assembly 20 via support arm76 and slider block 72. The distance between camera 44 and lens assembly20 is adjusted and maintained by a linear actuator 78 having a base 80and a shaft 82. An exemplary linear actuator, available from UltraMotion (Mattituck, N.Y.), is the “Bug” actuator, model numberD-A.083-AB-24501-2-2-K-B. Shaft 82 moves relative to base 80 whenactuator 78 is activated. Base 80 of actuator 78 is mounted to supportarm 56, while shaft 82 is attached to the underside of support arm 76.In this way, activating actuator 78 to focus the camera moves supportarm 76 (and thus moves camera 44) relative to support arm 56 (i.e.,relative to upper portion 24 of lens assembly 20). This arrangementallows for a secure, stable mounting of the lens in a manner where thelens magnification and camera focus can be separately adjusted.

As mentioned above, imaging system 40 includes chamber 46 for enclosingthe plate during imaging. The chamber may be as simple as a stage forholding the plate, or a partial enclosure into which a plate may bemoved into position for imaging. In preferred embodiments, however, thechamber will act to seal out undesired extrinsic light so it is nottransmitted by the lens to the camera. As shown in FIG. 4, chamber 46 issimply a cavity in support base 50 with an opening 84 for receiving amultiwell plate. Chamber 46 is light-tight, with the exception ofopening 84. Typically, when a plate is imaged, opening 84 is coveredwith a black cloth and/or the lights in the room are turned off to sealout undesired light. Light can also be sealed out by fitting opening 84with a light-tight door.

Camera 44 may be any suitable camera. To maximize the sensitivity of thesystem, however, a high-sensitivity cooled CCD is preferably employed.An exemplary cooled CCD camera is the 600 Series from SpectralInstruments (Tuscon, Ariz.) cooled with, e.g., liquid nitrogen or the“CRYOTIGER” cooling system (APD Cryogenics Inc, Allentown, Pa.). Imagesacquired with the CCD camera may be downloaded to a computer andanalyzed with any suitable image processing software, as is well knownin the art.

According to one embodiment of the invention, the imaging system furthercomprises a means for positioning a plate in the chamber. In a trivialexample, the means is simply the operator placing a plate onto a stageunder the telecentric lens. In preferred embodiments, however, suchmeans are automated, such as a conveyer belt plate delivery system or arobot for placing the plates under the lens for imaging.

An exemplary automated imaging system is shown in FIG. 5 as referencecharacter 90. Automated imaging system 90 is typically mounted on asupport surface, such as a table 92 having a top 94 which includes aplurality holes 96 (with only a few of holes 96 being shown forconvenience of illustration). The distance between each of holes 96 isknown so that various pieces of equipment may be attached to top 94,with the distance between the pieces of equipment known. An exemplarytable top which may be used with the invention is a TMC optical tabletop. Shown attached to table top 94 are lens support 42, lens assembly20, camera 44, and a robot 98. Table top 94 may further include one ormore racks 102, each of which contains a plurality of shelves 104 havingraised portions 106 for holding standard sample plates, as well as afluid dispensing station 108, and other equipment recognized by one ofskill in the art as being able to facilitate plate-based automatedlight-detection assays.

Robot 98 is designed to place or hold multiwell plates under thetelecentric lens for imaging. In a preferred embodiment, robot 98 isessentially identical to the robot described in co-owned, co-pendingU.S. patent application titled “Systems and Methods for Handling andManipulating Multi-Well Plates”, Ser. No. 08/937,139, filed Sep. 24,1997, herein incorporated by reference in its entirety.

The robot has an arm with a grasping mechanism which is configured tograsp a standard multi-well plate in a repeatable and predictable mannersuch that the location of the wells relative to the robot is known eachtime the plate is grasped. The grasping mechanism is configured suchthat each time a plate is grasped, it will be received at a preciselocation on the grasping mechanism so that the robot will know theprecise location of each of the wells. Because the location of the platerelative to the robot is precisely known, the robot may be used to holda multiwell plate in chamber 46 while an image is acquired (i.e., thereis no need to place the plate on a special stage inside chamber 46).

Referring now to FIGS. 6-8, an exemplary embodiment of a graspingmechanism 110 which is useful in connection with a robot will bedescribed. Grasping mechanism 110 comprises a frame member 112 which isconfigured to receive a standard sample plate or standard multi-wellplate 114. Multi-well plate 114 includes a plurality of wells 116 whichare arranged in a two dimensional array (for convenience ofillustration, only a selected number of the wells are shown).

Frame member 112 includes four raised edges 118 which serve as stopswhen plate 114 is forced against them. Edges 118 are arranged such thatthey will engage two sides of plate 114 when plate 114 is grasped. Aclamping arm 120 is pivotally attached to frame member 112 in a mannersuch that it may be forced against a corner of plate 114. As clampingarm 120 pushes against the corner, it forces plate 114 against edges 118to secure plate 114 to a precise location relative to frame member 112.

Edges 118 and clamping arm 120 are inwardly tapered as they approachframe member 112. In this way, when clamping arm 120 is pivoted againstthe corner, plate 114 will be forced downward against frame member 112.Hence, the location of plate 114 relative to frame member 112 will besecured in the X, Y and Z directions. In this manner, grasping mechanism110 can be used with a wide variety of standard sample plates havingstandard sized outer dimensions and formats since the location of eachwell can be repeatedly and precisely fixed relative to frame member 112.

Clamping arm 120 is pivotally attached to frame member 112 by an armshaft 124. To pivot clamping arm 120 relative to frame member 112, anair actuated piston 126 having a rod 127 in combination with a spring134 is employed. Piston 126 is attached to frame member 112 by a mountbracket 128, and spring 134 is attached to an arm post 122 on framemember 112. Between piston 26 and clamping arm 120 is a clevis 130 and alinkage 132. With such a configuration, spring 134 (which is in tension)causes clamping arm 120 to pivot about shaft 124 to apply a forceagainst the corner of plate 114 as previously described. To releaseclamping arm 120, piston 126 is actuated to causing rod 127 to retract.In this manner, clamping arm 120 is pivoted in an opposite direction torelease it from plate 114. As rod 127 of piston 126 retracts, spring 134expands to allow clamping arm 120 to pivot.

As best shown in FIG. 6, frame member 112 includes a patterned opening136. As described in greater detail hereinafter, patterned opening 136is adapted to allow frame member 112 to pass over a patterned shelf whengrasping plate 114.

Grasping mechanism 110 further includes an attachment member 138 whichallows grasping mechanism 110 to be operably attached to a robot.Mounted to attachment member 138 is a linear translator 140 which movesframe member 112 in an axial direction. For instance, as illustrated inFIG. 7, linear translator 140 is able to move frame member 112 parallelto an X axis. In this way, once plate 114 is positioned at a piece ofequipment, linear translator 140 may be employed to precisely move wells116 into alignment with a set of dispensing tips or syringes asdescribed in greater detail hereinafter.

Attached to two of the edges 118 is an alignment extension 142 whichtapers away from the inner surfaces of edges 118. In this way, whenframe member 112 is moved upward to grasp a plate, alignment extensions142 will assist in funneling the plate into alignment with edges 118.

Referring now to FIGS. 9A, 9B, 10A and 10B, robot 98 will be described.Robot 98 has a base member 148 and an arm 150. Pivotally attached to arm150 is grasping mechanism 110. As best shown in FIGS. 9A and 9B, arm 150comprises a shaft 150 a, an elbow 150 b, and a linkage 150 c.

Robot 98 is configured to move in a variety of directions to positionplate 114 at various locations. For example, as best shown in FIGS. 9Aand 9B, shaft 150 a is vertically movable in an up and down motion, i.e.along a Z axis. Shaft 150 a is also rotatable relative to base member148. Elbow 150 b is fixedly attached to shaft 150 a and rotates withshaft 150 a. Linkage 150 c is pivotable relative to elbow 150 b to allowa distal end of linkage 150 c having a turntable 151 to move in a radialdirection relative to base member 148. As previously described, framemember 112 is axially translatable relative to attachment member 138.One such robot that may be used with the invention is a ATM-205 robot,commercially available from Equipe Technologies. However, it will beappreciated that other robots may also be used. For example, theinvention employ a robot where radial motion is achieved by extensionand retraction of a piston.

Grasping mechanism 150 is attached to turntable 151 which is configuredto maintain a Y axis of plate 114 (see FIGS. 10A and 10B) parallel to aline between points A and B on arm 150 during vertical or rotationalmovement of shaft 150 a and/or during movement of linkage 150 c. In thisway, turntable 151 will rotate relative to linkage 150 c during movementof arm 150 to maintain the Y axis of plate 114 at a constant orientationrelative to the line between points A and B. With such a configuration,plate 114 will be properly oriented when moved to various locationssurrounding robot 98.

As shown in FIG. 5, attached to top 94 are a plurality of racks 102,each of which includes a plurality of patterned shelves 104. Shelves 104are configured to pass through patterned opening 136 of frame member 112(see FIG. 6) and which are provided to hold multi-well plates. In thismanner, robot 98 may be employed to grasp and remove a plate from one ofthe racks by positioning frame member 112 below one of the shelves 104which holds a plate that is to be removed. The robot aligns patternedopening 136 with the patterned self 104 and then lifts frame member 112vertically upward until frame member 112 lifts the plate from the shelf.As previously described, alignment extensions 142 (see FIG. 7) assist inpositioning plate 114 onto frame member 112. When plate 114 rests uponframe member 112, air piston 126 (see FIG. 7) is de-pressurized causingspring 134 to force clamping arm 120 against the corner of plate 114 aspreviously described. In turn, this forces plate 114 against edges 118to secure plate 114 to grasping mechanism 110. Robot 98 then moves plate114 to another one of the racks or another piece of equipment, e.g.,chamber 46, for imaging, processing, evaluation, or the like.

As shown in FIG. 11, raised portions 106 of shelf 104 assist in properlypositioning plate 114 on the shelf. For example, when placing a plate onshelf 104, grasping mechanism 110 is lowered until shelf 104 passesthrough patterned opening 136. At this point raised portions 106 guideplate 114 onto shelf 104 in a desired orientation.

V. Applications

As described above, the lens and system of the invention may be used ina variety of applications to image sample plates, such as multiwellplates and omni plates (plates having a single “well”). One example ofimaging an omni plate is in a lawn assay, where a lawn of cells, e.g.,bacterial cells, is plated in a solid growth medium, and the compoundsto be assayed, e.g., for activation of a luciferase reporter gene in thecells, are placed in contact with the growth medium at discrete points.Activation of the reporter is detected as a light signal from the plateat the location of the active compound.

The lens may also be used to image other substantially flat objects,such as round culture dishes, pieces of membrane or filter paper, gels,and sample arrays in other formats (e.g., arrays of immobilizedpolynucleotides). It can image light having wavelengths from about 350nm to over 750 nm, but is optimized for light between about 450 nm andabout 650 nm. Accordingly, suitable applications include calorimetricimaging, fluorescent imaging, luminescence imaging, phosphorescenceimaging and scintillation imaging.

The high level of sensitivity afforded by the lens and system describedherein allow for the imaging of extremely faint light signals, such asare generated in scintillation assays. Specifically, the presentinvention may be used to directly image light generated during ascintillation proximity assay (SPA; U.S. Pat. No. 4,568,649, hereinincorporated by reference). SPAs are homogeneous binding assays whichemploy a solid phase material containing a scintillant. Examples ofspecific embodiments include multiwell plates manufactured to containscintillant, and small beads (“SPA beads”) or “fluoromicrospheres”containing a scintillant.

While exemplary applications are described herein with respect to beads,it will be understood that they can be conducted using other solid phasescintillants suitable for use with SPA, such as multiwell plates withincorporated scintillant. In a typical application, the beads, availablefrom Amersham Life Science (Arlington Heights, Ill.), are derivatized(e.g., with a selected receptor) so as to bind specific molecules (e.g.,ligands for the selected receptor). When a radiolabelled molecule (e.g.,a radiaolabelled ligand) is bound to the bead, it stimulates thescintillant in the bead to emit light. Unbound radioactivity (e.g.,unbound or free radiolabelled ligand molecules) does not produce asignal, because the radioactive energy decays before it can reach a beadto produce a scintillation event. By way of example, if the radiolabelis tritium, the decaying tritium atom releases a beta particle. In anaqueous solution, the beta particle collides with water molecules anddissipates its energy within a mean distance of about 1.5 μm. Therefore,if the beta particle is emitted within a distance of about 1.5 μm of ascintillant molecule (in an SPA bead), it result in a scintillationevent. Otherwise, no light signal is detected. These principles aredescribed in more detail by, e.g., Bosworth, N. and Towers, P., Nature341:167-168 (1989) and by Udenfriend, S., et al., Anal. Biochem.,161:494-500 (1987).

Because SPAs are homogeneous assays, whereby unbound labeled moleculesdo not need to be removed from the assay mixture prior to detection ofbound molecules, they are widely used in the areas of clinical research,drug screening & discovery and pharmacology. Specific applicationinclude radioimmune assays, receptor binding assays (see, e.g.,Kienhuis, C. B. M., et al., J. Receptor Research 12:389-399 (1992)) andenzyme assays, including signal increase and signal decrease assays.Protocols for such assays are known in the art and can be obtained fromAmersham. For example, receptor binding assays can be conducted witheither solubilized or membrane-bound receptors. The receptor or themembrane in which it resides is typically immobilized onto the SPA beadand an appropriate ligand is radiolabelled (generally with either ¹²⁵Ior ³H). When the tracer binds to the receptor, the radioisotopestimulates the scintillant in the bead to emit light. By contrast, if anunlabelled ligand or competing drug (as in a drug-screening assay)replaces the tracer in the receptor binding site, less radioactivity isbound to the bead, and consequently less light is emitted. The presenceof molecules that are able to compete with the radiotracer for thereceptor may thus be detected.

Prior to the present invention, detection of light from SPA assays inmultiwell plates was typically performed using a scintillation counter,such as the “TOPCOUNT” from Packard Instrument Company (Meriden, Conn.).As detailed above, such detection methods are relatively slow and arenot presently suitable for use with high-density (e.g., 864-well)multiwell plates. Accordingly, presently-available methods forquantifying light from SPAs in multiwell plates is a bottleneck in highthroughput screening (HTS) applications. Example 1, below, describes howthis detection process can be streamlined through the use of atelecentric lens and system of the present invention. In the example,light from a scintillation proximity receptor binding assay in an864-well plate using the cholecystokinin B (CCKB) receptor is measuredin about 10 minutes using the lens and system of the invention.

When used to detect extremely low levels of light, as with SPA imaging,it is desirable to use a very low noise camera CCD camera as thedetection device. An exemplary camera suitable for suchhigh-sensitivity, low noise detection applications is the 600 Seriesfrom Spectral Instruments. This camera can be set to have a very slowread-out to limit the readout noise to less than 8 e—per readout. Toreduce the dark current, the CCD should be cooled, preferably to belowabout −80 degrees C. Such cooling may be accomplished using, forexample, liquid nitrogen or the “ CRYOTIGER” camera cooling system (APDCryogenics, Inc). A Spectral Imaging 600-series camera in conjunctionwith a “CRYOTIGER” has a dark current of less than 1 electron per hour,which together with the superior light collecting properties of thetelecentric lens of the invention, enable imaging of extremely faintlight signals, such as are generated in SPA experiments.

The following example illustrates but in no way is intended to limit thepresent invention.

EXAMPLE 1 Detection of Inhibition of CCK8 Binding to CCKb ReceptorAssayed by a Scintillation Proximity Assay

A COS cell line expressing the human cholecystokinin B (CCKb) receptor(CCKbR) was constructed using human CCKbR cDNA substantially asdescribed in Koller, K. J., et al., Anal. Biochem. 250:51-60 (1997).Membrane fragments from the CCKbR—expressing CHO cells were prepared asdescribed by Koller, et al., and were pre-incubated with wheatgermagglutinin-coated PVT SPA beads (Amersham). Approximately 0.25 mg of SPAbeads were combined with 2 μg or 1 μg of CCKbR membrane protein for eachwell of a 96-well or white 864-well plate, respectively. Unboundmembrane protein was removed from the beads by centrifugation (700Xg, 5min.) and the pellet (containing receptors bound to SPA beads) wasresuspended in assay buffer (20 mM HEPES, 1 mM EGTA, 118 mM NaCl, 5 mMMgCl₂, 5 mg/ml BSA, 4.7 mM KCl) to generate an assay cocktail.

50 or 100 pM ¹²⁵I-CCK8 (a highly selective non-peptide radioligand forthe CCKb/gastrin receptor, available from Amersham as catalog numberIM159) was added to the assay cocktail in the presence or absence ofvarious concentrations of unlabeled CCK8 to a final volume of 100 μl for96-well plates or 16 μl for 864-well plate. The plates were incubated atroom temperature overnight to reach equilibrium. The 864-well whiteplate was kept in the dark to allow for decay of plate phosphorescence,which would otherwise interfere with detection of scintillation events.

Following the overnight incubations, scintillation from the plates wasmeasured. Data from the 96-well plate were collected using the“TOPCOUNT” (Packard) following manufacturer's instructions. The 864-wellplate was imaged using a double telecentric lens system of the presentinvention. The plate was imaged twice for 10 minutes per exposure withthe Series 600 CCD camera from Spectral Imaging cooled using the“CRYOTIGER” system as described above.

The image data were analyzed using the IPLab Spectrum 3.1 program(Scanalytics, Incorporated, Fairfax, Va.). The two images were overlaidand the minimum value for each corresponding pixel was used to generatea final image (to correct for background noise due to cosmic rays). Darknoise generated by the CCD itself, corresponding to the temperature atthe time the images were captured, was also subtracted.

An exemplary image generated as described above is shown in FIG. 12. Theexperiment was conducted with different amounts of CCKbR membraneprotein (indicated along the left side) and at two different ¹²⁵I-CCK8concentrations (indicated along the top). At each CCKbR membrane proteinand ¹²⁵I-CCK8 concentration, the assay cocktail was incubated induplicate with 16 different concentrations of unlabeled CCK8. These datawere then used to generate binding inhibition curves, shown in FIG. 13.As can be appreciated from FIG. 13, increasing amounts of receptorresulted in a rightward shift of the inhibition curve and correspondingIC₅₀ values. The IC₅₀ values are plotted as a function of CCKbR membraneprotein concentration in the inset. The data indicate that a receptormembrane concentration of about 1 μg per well is optimal for drugscreening studies with CCKbR and 125I-CCK8. The inhibition curves andcorresponding IC₅₀ values determined using the 96-well plates and“TOPCOUNT” were in accord with those determined from the images asdescribed above. Taken together, the data demonstrate that an imagingsystem of the invention, using a telecentric lens of the invention, iscapable of capturing image data from a bead-based SPA assay in ahigh-density multiwell plate in a reasonable period of time. Further,the results show that the image data are uniform across the plate, andcan thus be quantitatively analyzed, e.g., to generate bindinginhibition curves and corresponding IC₅₀ values.

While the invention has been described with reference to specificmethods and embodiments, it is appreciated that various modificationsand changes may be made without departing from the invention.

It is claimed:
 1. A multiple element doubly telecentric lens for imaginga substantially flat object onto an image plane, said lens having thefollowing characteristics: (i) between 12 and 16 lens elements; (ii) anumerical aperture of between about 0.5 and 0.6; (iii) less than about5% vignetting; (iv) less than about 0.05% distortion; (v) amagnification of between about −0.20 and about −0.25; and (vi) a fieldof view of between about 4 and about 6 inches in diameter.
 2. Atelecentric lens of claim 1, having 12 lens elements.
 3. A telecentriclens of claim 1, for imaging a multiwell plate having square wells ontoa CCD pixel array, said lens having a magnification that results in eachwell of said plate mapping to an integer number of pixels in said CCDarray.